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This page outlines the diagnostic approaches to NCL
Diagnostic strategies vary according to patient age. Investigations are changing, such as the new technologies capable of testing for many genes. Nevertheless, it can still be helpful to apply more established methods to confirm diagnosis.
Enzyme analysis
The enzyme activities of the CLN1/PPT1, CLN2/TPPI and CLN10/CTSD enzymes can be measured. Low levels are definitive of NCL caused by mutations in these genes, even in cases of adult onset. These enzyme tests can be carried out using blood, skin biopsy or saliva as the source of cells and care must be taken to use the most specific assays possible, especially for cases of later onset. It should be a routine matter to perform these enzyme assays on all cases of NCL, whatever the age of onset, to confirm NCL caused by mutations in these genes. Cases with apparently normal enzyme levels in blood samples but clinical features consistent with disease caused by mutations in CLN1/PPT1, CLN2/TPP1 or CLN10/CTSD should be followed up with enzyme and EM analysis of fibroblasts obtained via skin biopsy.
Mutation analysis
The identification of a mutation in both copies of an NCL gene is definitive for diagnosis of NCL and of NCL type. This also allows accurate carrier and prenatal testing. Even when enzyme tests are confirmatory for diagnosis, mutation analysis is routinely performed since knowledge of the underlying mutations is usually required. Defining the mutations present is the only way to reliably assign NCL type. For some patients no mutations in any known NCL gene will be found, either because they carry mutations in a gene that has not yet been identified, or because their mutations are undetectable by the techologies used (eg they may be in the gene promoter or intronic regions), or because they do not have NCL. For such cases, the contribution of samples for research will aid future gene identification.
Electron microscopy
Cells from patients with NCL accumulate storage material which can be examined ultrastructurally using electron microscopy. Various patterns of these NCL-specific structures are recognised. Diagnosis, particularly of variant types, can be aided by electron microscopy. The tissue best suited to this is a skin biopsy. A 3 mm punch biopsy deep enough to include sweat glands is ideal. Skin contains a wealth of diverse types and therefore provides a higher diagnostic yield than lymphocytes. The most important cell type in the skin for diagnosis is the secretory eccrine sweat gland epithelial cell. It is good practice to require observation of storage in at least two cell types to reach a diagnosis of NCL. Fibroblast cells in general do not exhibit evidence of storage, except for GRODs arising from mutations in CLN1. Granules from mast cells are a frequent source of misdiagnosis. The biopsy should be taken from a site where sweat glands are present (eg the inside of the upper arm but not the axillary region, or the forearm) and ideally the biopsy should be transferred to 2.5% buffered glutaraldehyde for preservation or if this is not practical 10% buffered formaldehyde. Alternatively examination of circulating lymphocytes may also be of use and is minimally invasive. Lymphocytes should be isolated as a buffy coat and well fixed. Care must be taken to distinguish between NCL-specific material and normally ocurring similar structures, particularly after a viral illness. Use of peripheral blood alllows reliable and specific diagnosis of NCL and can readily distinguish between NCL types. In the past rectal biopsy and tissues such as conjuntiva or skeletal muscle were used but this is not so common now, and inclusions may take on a different pattern in muscle fibes complicating diagnosis of NCL type. For diagnosis of adult onset NCL, a brain biopsy might be considered since storage may not be found in a skin biopsy or circulating lymphocytes. For a brain biopsy, a small piece should be snap frozen in liquid nitrogen, and the main portion divided for routine analysis (preserved in buffered formaldehyde) and ultrastructural investigation (buffered glutaraldehyde). This will provide ample opportunity to achieve all investigations.
Light microscopy
Lymphocytes from patients juvenile CLN3 disease contain vacuolations that can be readily seen by light microscopy of a stained peripheral blood film, particularly in the trails. Other NCLs very rarely show vacuolated lymphocytes. Vacuolations are found in lymphocytes from patients with some other lysosomal storage disorders, and care must also be taken not to confuse vacuolations with swollen mitochondria. Rectal biopsy containing nerve cells (not obtained if suction biopsy is used), as cryostat sections of frozen samples, was formerly used to determine if neuronal storage was present and to define which cell types were involved, using an acid phophatase reaction, staining with Sudan black and examination. Autofluorescence could also be detected, but care had to be taken to distinguish between normal age pigment and the NCL-specific pigment, which could be done with a good combination of filters. Light microscopic findings must be corroborated by electron microscopy and/or mutation analysis and/or enzyme analysis.
Cell lines
Establishing a cell line from a fresh skin biopsy placed in tissue culture medium is a simple procedure. An alternative approach is by transformation to make lymphoblast cell line from a blood sample. In terms of future diagnostic requirements this avoids the need for repeated samples to be taken from the patient. A cell line allows an unending supply of material that can be used to make DNA or RNA and provides cells suitable for biological research (for cell biology and microscopic studies). Establishing a cell line is recommended for cases of unusual disease course or when it has not been possible to determine the exact type of NCL, or when a rare mutation has been found.
Prenatal diagnosis
Diagnostic tests can be carried out prenatally to establish whether a baby is affected or is healthy (and whether a carrier or not) in a family that has already had an affected child or when both parents are known to be carriers of a mutation in the same NCL gene. There are three main ways that this can be done, each requiring access to chorionic villus cells (CVS - chorionic villus sampling) from the placenta: 1) Mutation analysis in families in whom the genetic defect has been identified; 2) Enzyme analysis in families already shown to have an enzyme deficiency; 3) Identification of typical inclusions by electron microscopy in families in whom there is no enzyme deficiency and the gene has not been identified. The procedure is usually performed between 10-15 weeks of a pregnancy. If the genetic basis within a family is known, pre-implantation genetic diagnosis (PGD) combined with in-vitro fertilisation (IVF) can be offered to identify embryos that will not be affected by NCL disease.